Project Summary/Abstract High doses of ionizing radiation causes massive cell death and severe damage to the gastrointestinal (GI) tract which is referred to as GI syndrome. The GI syndrome is a common and severe complication in cancer patients undergoing radiotherapy or chemotherapy, but the cellular and molecular mechanisms are unclear and there is no effective therapy. Absent in melanoma 2 (AIM2) forms an inflammasome with DNA and the adapter protein ASC to initiate caspase-1 activation, secretion of inflammatory cytokines and a form of cell death known as pyroptosis. However, the functional role of AIM2 in the GI syndrome was not appreciated. Our recent studies using mouse models of subtotal body irradiation (SBI) induced GI syndrome showed (i) Aim2-/-, Asc-/- Casp-1-/- and mice had reduced GI syndrome and mortality. (ii) Remarkably, nuclear AIM2 bound DNA at sites of DNA double-strand breaks (DSBs) in a complex with ?-Histone 2AX foci to initiate inflammasome activation in response to radiation, suggesting AIM2 may monitor genomic instability through sensing DSBs. The rationale to our approach is based on the scientific premise supporting AIM2 as a DNA sensor in detecting DSB initiated signals to facilitate cell death in GI syndrome. Thus, we hypothesize that AIM2 sensing DSBs signal is a newly identified core feature of the sensing of genomic integrity and inflammasome coordinated pyroptotic IEC death. We will therefore test following three specific aims: Aim1: Determine the mechanism of cell death mediated by the AIM2 inflammasome in response to DNA damage; 1). Determine the contributions of different forms of cell death to GI syndrome. 2). Identify the dynamic subcellular localization of AIM2 inflammasome components during both steady state and activation by radiation. 3). Test the efficacy of a clinically relevant Caspase-1 inhibitor VX-765 to mitigate GI syndrome in APCmin mouse model. Aim2: Determine the intersection of the AIM2 inflammasome with canonical DSB response; 1) Investigate the DSB response in Aim2-, Asc- and Gsdmd-deficient mice. 2). Identify the role of the AIM2 inflammasome in the canonical DNA damage response. 3). Characterize the dynamic interaction of AIM2 with the DSB repair proteins of the ATM/DNA-PK/p53 pathway. Aim3: Identify novel AIM2 partner(s) as a potential radio-/chemo-sensitizing target in GI syndrome. 1). Define the molecular composition of the AIM2 interactome that responds to radiation. 2). Determine the novel genetic AIM2 interactome in the control of radiation-induced DSB response and cell death in vitro through genome-wide CRISPR/cas9 library screening. 3). Validate and determine the functions of the novel AIM2 partner(s) in GI syndrome both in vitro and in vivo. Collectively, our proposed research will elucidate the novel regulatory mechanisms underlying the GI syndrome, and lay the foundation for developing novel therapeutics for GI syndrome by manipulation of DSBs sensing AIM2 activation.